title: The role of the disulfide bridge in the stability and structural integrity of ovalbumin evaluated by site-directed mutagenesis.
abstract: To provide a molecular explanation of the role of the disulfide (SS) bridge in the thermostability and structural integrity of ovalbumin (OVA), we prepared SS-mutated OVAs in which SS-forming residues were replaced by Ala or Ser (C73A, C73S, C120A, and C73/120A), and compared the conformation, thermostability, susceptibility to elastase, and formation of heat-stable OVA (S-OVA) with those of the wild-type. The circular dichroism (CD) and tryptophan fluorescence spectra revealed that the SS-mutated OVAs assumed a native-like conformation similar to the wild-type. The thermal denaturation temperature for the SS-mutated OVAs was significantly lower than that for the wild-type. C73S, C120A, and C73/120A mutants converted to S-OVA on alkaline treatment. Analyses for elastase digestion fragments showed that a non-native SS bridge was generated in all SS-mutated OVAs, but non-native SS-pairing did not contribute to thermostability. Hence, we concluded that the presence of the original SS bridge in OVA contributes to conformational stability but is not directly related to the conversion to S-OVA.
authors.affiliation: Department of Biological Chemistry, Yamaguchi University, Yamaguchi, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ishimaru
authors.forename: Kazunari
authors.initials: K
authors.lastname: Ito
authors.forename: Miho
authors.initials: M
authors.lastname: Tanaka
authors.forename: Syunpei
authors.initials: S
authors.lastname: Tanaka
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Matsudomi
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010047:Ovalbumin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012694:Serine; D010047:Ovalbumin; D010196:Pancreatic Elastase; D000409:Alanine